Process for producing vitamin B6

ABSTRACT

Disclosed is a mutant of a recombinant microorganism of the genus  Sinorhizobium  capable of producing vitamin B 6  having a recombinant plasmid with pdxJ gene that acquired histidine requirement of glycine resistance, or its combination thereof.

This application is the National Stage of International Application No.PCT/EP2003/010296, filed Sep. 16, 2003.

The present invention relates to a novel microorganism and a process forpreparing vitamin B₆ by using the same.

“Vitamin B₆” as used in the present invention includes pyridoxol,pyridoxal, and pyridoxamine. Vitamin B₆ is a vitamin indispensable tohuman beings or other animals and used as a raw material of medicines oras feed additives. As a process for preparing vitamin B₆ in afermentation method, a process for preparing vitamin B₆ using amicroorganism derived from the genus Sinorhizobium (also known asRhizobium) (EP 765,938) is known. But it is necessary to construct a newmicroorganism with a higher yield of vitamin B₆ and lo to develop animproved industrial fermentation process which can produce vitamin B₆with sufficiently high production efficiency using said microorganism.

According to the present invention, it is possible to produce vitamin B₆more efficiently than the previous process. The present inventors firstconstructed a recombinant micro-organism of the genus Sinorhizobiumcapable of producing vitamin B₆ incorporated with a recombinant plasmidcomprising a vector containing pyridoxol 5′-phosphate synthase gene(referred to as pdxJ hereinafter). This recombinant microorganism showedan increased production of vitamin B₆. This recombinant microorganismwas further mutated to acquire a phenotypic property of histidinerequirement, or glycine resistance. Such a mutant microorganism showsfurther increased productivity of vitamin B₆. A mutant which acquiredthe above mentioned two phenotypic properties simultaneously showsdrastically increased productivity of vitamin B₆, and that vitamin B₆can advantageously be produced in the culture broth by cultivating themicroorganism, and can be recovered therefrom in a desired purity.

The present invention provides a mutant of a recombinant microorganismof the genus Sinorhizobium capable of producing vitamin B₆ having arecombinant plasmid with pdxJ gene that acquired a phenotypic propertyof histidine requirement or glycine resistance, or combination of thephenotypic properties thereof.

Another object of the present invention is to provide a process forpreparing vitamin B₆ which comprises cultivating said microorganism in aculture medium and collecting the produced vitamin B₆.

As a parent strain for preparing a microorganism in the presentinvention, any strains belonging to the genus Sinorhizobium capable ofproducing vitamin B₆ can be used, and a microorganism belonging to thegenus Sinorhizobium may be isolated from natural sources, or may bepurchased from culture collections. S. meliloti IFO 14782 (DSM 10226) ispreferable to the present invention. A microorganism capable ofproducing large amount of vitamin B₆ can be constructed as describedbelow.

(1) Preparation of S. meliloti IFO 14782 Harboring Recombinant Plasmidwith pdxJ Gene

[A] Construction of Expression Plasmid for pdxJ

“pdxJ” referred to herein means the gene encoding an enzyme catalyzingsynthesis of pyridoxol 5′-phosphate from 1-deoxy-D-xylulose 5-phosphateand aminoacetone 3-phosphate. A pdxJ gene derived from microorganismbelonging to the genus Sinorhizobium is preferable. For example, a DNAof pdxJ derived from S. meliloti IFO 14782 can be cloned in thefollowing manner. The primers for polymerase chain reaction (referred toas PCR hereinafter) are synthesized in accordance with the DNA sequenceof pdxJ in a DNA data-base of S. meliloti strain 1021, and which containrestriction enzyme recognition site at the 5′ end of each primer. ThepdxJ gene can be amplified by PCR using the primers and chromosomal DNAof S. meliloti IFO 14782. Amplified pdxJ is ligated into a vectorreplicable in Escherichia coli such as available pUC series or pBRseries. A plasmid, wherein pdxJ is inserted, can be selected by agarosegel analysis of the plasmid digested with endonuclease, and the sequenceof amplified region can be ascertained with a DNA sequencer.

As a vector for expression of PdxJ protein in E. coli, a vector can beremodeled into a new plasmid, which has a promoter functioning in E.coli such as ptac, ptrp, plac, or ptrc followed by restriction enzymerecognition sequence. A plasmid expressing the PdxJ protein in E. colican be provided by inserting the thus-obtained pdxJ into a thus-obtainedexpression plasmid, which encodes pdxJ under control of a promoter.

As a vector for expression of PdxJ protein in S. meliloti, a broad-hostrange vector, such as pVK100, pRK290, pLAFR1 or RSF1010, can be used. Aplasmid expressing PdxJ protein in S. meliloti can be provided byinserting a DNA fragment encoding a promoter functioning in S. meliloti,such as ptac, plac, ptrc, pS1 (promoter of small ribosomal subunit of S.meliloti), or pNm (promoter of neomycin resistant gene) and pdxJ into abroad-host range vector.

The procedure for constructing recombinant vectors can be performedaccording to standard techniques known in the fields of molecularbiology, bioengineering, and genetic engineering.

[B] Introduction of Recombinant Plasmid with pdxJ into S. meliloti IFO14782

A plasmid encoding pdxJ of S. meliloti can be transformed to E. coliaccording to standard techniques known in the fields of molecularbiology, bioengineering, and genetic engineering.

A broad-host range plasmid encoding pdxJ can be introduced into S.meliloti IFO 14782 by tri-parental mating in the following manner. S.meliloti as a recipient strain, E. coli harboring helper plasmid as ahelper strain, and E. coli harboring donor plasmid as a donor strain arecultivated separately and mixed together. After mixed cultivation onplate, S. meliloti receiving a recombinant plasmid can be selected onagar plate containing appropriate antibiotics. The plasmids of coloniesgrown on the plates are examined by endonuclease digestion.

(2) Preparation of Mutant Endowed with Amino Acid Requirement Induced byMutagenesis with N-methyl-N′-nitro-N-nitrosoguanidine (referred to asNTG hereinafter)

Pyridoxol in S. meliloti IFO 14782 is known to be synthesized by ringclosure of two precursors, 1-deoxy-D-xylulose and 4-hydroxy-L-threonine[Tazoe et al., J. Biol. Chem. 275:11300-11305 (2000)]. In general,accumulation of amino acids synthesized by a branched pathway isreported to be greatly enhanced by induction of amino acid requirement.Thus it is conceivable to isolate amino acid requiring mutants to gethigher vitamin B₆ producer. S. meliloti IFO 14782/pVKP601 prepared in(1) [B] is subjected to NTG mutagenesis to produce mutants producingpyridoxol more in the culture broth by induction of amino acid requiringmutants. Sinorhizobium (Ensifer) meliloti IFO 14782/pVKP601 wasdeposited on Feb. 29, 2008 under deposit number DSM 21235 at the DSMZ(Deutsche Sammlung von Mikroorganismen und Zeikulturen GmbH),Inhoffenstraβe 7 B, 38124 Braunschweig, Germany under the provisions ofthe Budapest Treaty. Cells of the strain are treated with NTG. Aftertreatment, a restorative cultivation is carried out and the resultingculture is plated out on agar medium. To isolate mutants requiring aminoacid, the growth of colonies is tested on agar of inorganic nitrogensalt medium containing vitamins and nucleic acids. From the test,colonies requiring amino acid can be selected, and a vitamin B₆ highproducer may be selected by testing productivity of vitamin B₆ in thefermentation. The strain S. meliloti PY-C341-K1 is one of the objectivemutants in this present invention. Sinorhizobium (Ensifer) melilotiPY-C341-K1 was deposited on Feb. 29, 2008 under deposit No. DSM 21236 atthe DSMZ (Deutsche Sammlung von Mikroorganismen und Zeikulturen GmbH),Inhoffenstraβe 7 B, 38124 Braunschweig, Germany under the provisions ofthe Budapest Treaty.

(3) Preparation of Mutants Endowed with Glycine Resistance Induced byMutagenesis with NTG

Biosynthesis of vitamin B₆ in S. meliloti IFO 14782 is well-known asdescribed in (2). Pyridoxol is synthesized from a sugar and an aminoacid precursors, and the latter precursor is from glycolaldehyde andglycine. Glycine is not only one of vitamin B₆ precursors but also astrong inhibitor to the growth of the strain. Accordingly, induction ofglycine resistant mutant leads to enhancement of production of vitaminB₆. To isolate glycine resistant mutants, minimal inhibitoryconcentration of glycine against the strain should be examined on anappropriate medium because the inhibition strength was different intested medium. Thus, strain PY-C341-K1 obtained in (2) is subjected toNTG mutagenesis to produce glycine resistant mutants. Cells of thestrain are treated with NTG in a similar manner as described in (2).After treatment, a restorative cultivation is carried out and theresulting culture is plated out on an agar medium. To isolate glycineresistant mutants, the cell suspension is spread onto plates of agarmedium containing appropriate concentration of glycine. Afterincubation, colonies resistant to glycine may be selected on agar mediumcontaining glycine.

The microorganisms obtained in the present invention are incubated in amedium containing an assimilable carbon source, a digestible nitrogensource, an inorganic salt, and other nutrients necessary for theirgrowth. As a carbon source, for example, glucose, fructose, lactose,maltose, galactose, sucrose, starch, dextrin, or glycerol may beemployed. As a nitrogen source, for example, peptone, corn steep liquor,soybean powder, yeast extract, meat extract, ammonium chloride, ammoniumsulfate, ammonium nitrate, urea, or their mixture thereof may beemployed. Further, for trace elements, sulfates, hydrochlorides, orphosphates of calcium, magnesium, zinc, manganese, cobalt, and iron maybe employed. And, if necessary, conventional nutrient factors, atrapping agent of phosphate ion, or an antifoaming agent, such asmagnesium carbonate, aluminum oxide, allophane, animal oil, vegetableoil, or mineral oil can also be added supplementary in a fermentationmedium.

The pH of the culture medium may be about 5.0 to 9.0, preferably 6.5 to7.5. The cultivation temperature may be about 10° C. to 40° C.,preferably 25° C. to 35° C. The cultivation time may be about 1 day to15 days, preferably 2 days to 9 days.

In the cultivation, aeration and agitation usually give favorableresults.

After the cultivation, vitamin B₆ produced also may be separated fromthe culture broth and purified. For this purpose a process generallyused for extracting a certain product from the culture broth may beapplied by utilizing various properties of vitamin B₆. Thus, forexample, the cells are removed from the culture broth, the desiredsubstance in the filtrate is absorbed on active carbon, then eluted andpurified further with an ion exchange resin. Alternatively, the culturefiltrate is applied directly to an ion exchange resin and, after theelution, the desired product is recrystallized from mixture of alcoholand water.

The microorganisms used in the present invention include all the mutantstrains of genus Sinorhizobium capable of producing vitamin B₆ having arecombinant plasmid with pdxJ gene that acquired phenotypic property ofhistidine requirement, or glycine resistance, or combination of theirphenotypic properties thereof. Among the strains of genus Sinorhizobium,a particularly preferred strain is S. meliloti PY-EGC1, which wasdeposited on Sep. 17, 2002 under deposit number DSM15209 at the DSMZ(Deutsche Sammlung von Mikroorganismen und Zeilkulturen GmbH) inGöttingen (Germany) under the Budapest Treaty.

The present invention will be explained more in detail by referring tothe following examples; however, it should be understood that thepresent invention is not limited to those particular examples.

In the Examples, the amount of vitamin B₆ produced in culture broth canbe assayed by the turbidity method with Saccharomyces carlsbergensisATCC 9080 [Osbone and Voogt, The Analysis of Nutrients in Foods,Academic Press, London, 224-227 (1978)], and vitamin B₆ derivatives suchas pyridoxol, pyridoxal, and pyridoxamine in a fermentation broth can beseparately quantified by high pressure liquid chromatography (referredto as HPLC hereinafter).

EXAMPLE 1 Preparation of S. meliloti IFO 14782/pVK601

(1) Cloning of pdxJ of S. meliloti IFO14782

To amplify pdxJ of S. meliloti IFO 14782 using PCR method, the followingtwo primers were synthesized according to the DNA sequence of pdxJ(2249854-2250606, complement) in the genome database of S. melilotistrain 1021 (Accession No. NC_(—)003047): primer A (SEQ ID NO:1) withrestriction enzyme NdeI recognition sequence including start codon ofpdxJ and primer B (SEQ ID NO:2) with PstI site just after stop codon ofpdxJ. Chromosomal DNA was extracted from the cells grown in a medium(referred to as LBMC hereinafter) composed of 1% Bacto Tryptone (BectonDickinson Microbiology systems, MD, USA), 0.5% Bacto Yeast extract(Becton Dickinson Microbiology systems, MD, USA), 0.5% NaCl, 0.061%MgSO₄.7H₂O, and 0.036% CaCl₂.2H₂O with QIAGEN genomic-tips (QIAGEN GmbH,Germany).

PCR was performed using advantage-HF PCR kit (CLONTECH Laboratories,Inc. CA, USA). 100 μl of reaction mixture contained 10 ng of chromosomalDNA of S. meliloti IFO 14782, 50 pmol of the two primers, 10 μl of 10×HF dNTP mix, 10 μl of appended 10× HF PCR reaction buffer, and 2 μl of50× advantage-HF polymerase mix. The reaction conditions were asfollows; holding at 94° C. for 3 min., 4 cycles of 30 sec at 98° C., 1min at 53° C., 1 min at 72° C., 20 cycles of 30 sec at 98° C., 1 min 68°C., and holding at 72° C. for 10 min. 10 μl of reaction mixture wassubjected to agarose gel on 1% (w/v) gels, and a DNA band of 770 bp wasrecovered from the gel with QIAEXII (QIAGEN GmbH, Germany). The fragmentwas ligated to pUC18, which was digested with SmaI and dephosphorylatedwith alkaline phosphatase, by SureClone ligation kit (AmershamBiosciences Corp., NJ, U.S.A.).

The thus obtained ligation mixture was transformed into E. coli JM109competent cells (Takara Bio Inc., Shiga, Japan) and plated on plates ofa medium composed of 1% Bacto Tryptone, 0.5% Bacto Yeast extract, and0.5% NaCl (referred to as LB hereinafter) containing 100 μg/ml ofampicillin (referred to as Amp). Plasmids of colonies grown on theplates were prepared with Automatic DNA Isolation System PI-50 (KuraboIndustry Ltd., Japan). By analysis of the plasmid with restrictionenzyme, a recombinant plasmid pSHT56, wherein pdxJ was the samedirection as lacZ gene on pUC18, was obtained. pSHT56 was prepared fromE coli JM109 harboring pSHT56 with QIAGEN plasmid Midi kit (QIAGEN GmbH,Germany). The DNA sequence of pdxJ in the plasmid was ascertained withan ALF DNA sequencer (Amersham Biosciences Corp., NJ, U.S.A.) and it wasidentical with that of a genome database of S. meliloti strain 1021.

As a vector for expression of pdxJ in E. coli, pUC 18 was remodeled intopUC-trc2, which has trc promoter region of pTrc99A (Amersham BiosciencesCorp., NJ, USA) followed by NdeI recognition sequence. pUC-trc2 wasprepared from E. coli JM109 harboring pUC-trc2 with QIAGEN plasmid Midikit. To give an expression plasmid for pdxJ in E. coli, pUC-trc2 wasdigested with NdeI, and a 2.5-kb fragment was recovered from agarose geland dephosphorylated with alkaline phosphatase (Takara Bio Inc., Shiga,Japan). On the other hand, pSHT56 was cleaved with NdeI, subjected toagarose gel and resulting 1-kb fragment was recovered from the gel withQIAEXII. The recovered 1-kb fragment was ligated to prescribed 2.5-kbfragment of pUC-trc2 with ligation kit (Takara Bio Inc., Shiga, Japan).E. coli JM109 was transformed with thus-obtained ligation mixture andplated on LB plates containing 100 μg/ml of Amp. Plasmid of a colonygrown on the plate was prepared with Automatic DNA Isolation SystemPI-50. By analysis of the plasmid with restriction enzyme, a recombinantplasmid pSHT57, wherein pdxJ was the same direction as trc promoter, wasobtained (FIG. 1). pSHT57 was prepared from E. coli JM109 harboringpSHT57 with QIAGEN plasmid Midi kit.

(2) Construction of Expression Plasmid for pdxJ in S. meliloti IFO 14782

To construct an expression vector in S. meliloti IFO 14782, pVK100 wasused, which is reported to be a broad host range vector, IncP-1 type,and replicable in S. meliloti. pVK100 was prepared from E. coliHB101/pVK100 with QIAGEN plasmid midi kit, and digested with HindIII,blunt-ended by blunting kit (Takara Bio Inc., Shiga, Japan) anddephosphorylated with alkaline phosphatase. pSHT57 was digested withBamHI and KpnI. Resulting 875-bp fragment, which contained trc promoterand pdxJ, was recovered from agarose gel, blunt-ended by blunting kit,and ligated to prescribed pVK100 with ligation kit. E. coli HB101competent cells (Takara Bio Inc., Shiga, Japan) was transformed with theobtained ligation mixture and plated on LB plates containing 10 μg/ml oftetracycline (referred to as Tc hereinafter). Plasmids of colonies grownon the plates were prepared with Automatic DNA Isolation System PI-50.By analysis of the plasmid with restriction enzyme, a recombinantplasmid, pVIK601, wherein trc promoter and pdxJ were the oppositedirection against kanamycin resistant gene on, was obtained (FIG. 1).

(3) Complementing E. coli AT3208, pdxJ Mutant

pSHT57 was transformed into pdxJ mutant, E. coli AT3208 (purchased fromE. coli Genetic Stock Center, Yale Univ., U.S.A.). All Amp resistanttransformants grew on vitamin B₆-free EMM plate composed of 10 g ofglucose, 8 g of vitamin-free casamino acid (Becton DickinsonMicrobiology systems, MD, U.S.A.), 2.5 mg of MnSO₄.5H₂O, 125 mg ofMgSO₄.7H₂O, 125 mg of CaCl₂.2H₂O, 425 mg of KCl, 250 μg FeCl₃.6H₂O, 250μg of thiamin hydrochloride, 8 μg of biotin, 15 g of Bacto agar (BectonDickinson Microbiology systems, MD, U.S.A.) per liter (pH 6.8) whereaspdxJ mutant, E. coli AT3208, did not grow on the plate. This indicatedthat cloned pdxJ worked as PdxJ.

(4) Introduction of pVK601 into S. meliloti IFO 14782

pVK601 was introduced into S. meliloti IFO14782 by tri-parental matingas described below. S. meliloti IFO14782 as a recipient strain wasinoculated in 5 ml of liquid LBMC medium and incubated with shaking at30° C. at 140 rpm for 16 hours. 400 μl of the culture were transferredinto the fresh same medium and incubated further for 6 hours. E. coliHB101 harboring pRK2013 (Km^(r); IncP tra⁺, ColEI ori)(ATCC 37159) as ahelper strain was inoculated in 5 ml of liquid LB medium containing 50μg/ml of kanamycin and incubated with shaking at 37° C. at 140 rpm for16 hours. 100 μl of the culture were transferred into the fresh samemedium and incubated further for 6 hours. E. coli HB101 harboring pVK601was inoculated in 5 ml of liquid LB medium containing 10 μg/ml of Tc andincubated with shaking at 37° C. at 140 rpm for 16 hours. 100 μl of theculture were transferred into the fresh same medium and incubatedfurther for 6 hours. Each strain was harvested and cells were mixed at1:1:4 (v/v/v) ratio. The mixture was put on a nitrocellulose filterplaced on LBMC agar plate. After this plate was incubated for 20 hoursat 30° C., cells on the filter were scratched and suspended insterilized 0.85% NaCl solution. The suspension was diluted appropriatelyand spread on LBMC plates containing 20 μg/ml nalidixic acid (to selectfor S. melilot IFO14782) and 10 μg/ml Tc (to select for pVK601). Afterincubation of these plates at 30° C. for 5 days, colonies grown on theplates were picked up and cultured for plasmid extraction by QIAGENplasmid mini kit (QIAGEN GmbH, Germany). Thus-obtained plasmid DNA withtreatment of endonuclease showed an identical pattern to pVK601 onagarose gel. From this result, one colony in the tested colonies wasselected as S. meliloti IFO 14782/pVK601.

EXAMPLE 2

A loopful of S. meliloti IFO14782/pVK601 and the parent, S. melilotiIFO14782, grown on a LBMC agar plate at 30° C. for 48 hours wasinoculated to tubes containing 8 ml of a seed medium (referred to as SMhereinafter) composed of 1% glucose, 1% corn steep liquor (OjiCornstarch Co., Ltd., Tokyo, Japan), 0.2% Bacto yeast extract, 0.05%MgSO₄.7H₂O, 0.001% MnSO₄.5H₂O, and 0.001% FeSO₄.7H₂O (pH 7.0) and thenthe tubes were shaken on a reciprocal shaker (275 rpm) at 30° C. Aftershaking for 19 hours, each 4 ml of the cultures was transferred to a 500ml flask with two baffles containing 200 ml of a production medium(referred to as PM hereinafter) composed of 6% glucose, 3% corn steepliquor, 0.8% Bacto yeast extract, 0.35% NH₄Cl, 0.05% MgSO₄.7H₂O, 0.025%MnSO₄.5H₂O, 1% Allophosite (Shinagawa Chemicals Co., Ltd., Tokyo, Japan)and 0.025% Actocol (pH 6.8) and shaken on a rotary shaker (180 rpm) at30° C. After cultivation for 7 days, contents of vitamin B₆ in thesupernatant of each culture broth were quantified by high pressureliquid chromatography (referred to as HPLC hereinafter) and producedvitamin B₆ was calculated by the internal standard method with4′-deoxypyridoxol as described below. To prepare the samples for HPLC,100 μl of the solution containing 100 mg/l of 4′-deoxypyridoxol asinternal substance was added to 400 μl of the standard solutions ofpyridoxol and the supernatant from the culture broth, and then themixture was put on the following column. The analytical conditions wereas follows: column, Capcell pak C18 SG120 (4.6×250 mm) (Shiseido Co.,Ltd., Tokyo, Japan); mobile phase, 0.1 M sodium perchlorate, 0.1 Mpotassium phosphate, and 2% acetonitrile (pH 3.5); column temperature,25-26° C.; flow rate, 1.0 ml/min; and detector, ultraviolet (UV) (at 292nm). As a result, S. meliloti IFO14782 /pVK601 produced 119 mg ofpyridoxol per liter and was about 1.34 times higher than the parent,strain IFO 14782.

EXAMPLE 3

S. meliloti IFO14782/pVK601 obtained in Example 1 was cultured in aflask containing LBMCG containing 5 μg/ml of Tc for 17 hours at 30° C.,and the cell suspension of the strain was prepared. A tube containing 5ml of the reaction mixture composed of 150 μg/ml of NTG and 1.6 ×10⁹cells per ml in 50 mM Tris-HCl buffer (pH 8.0) was incubated with areciprocal shaking (275 rpm) for 30 min at 30° C. The cells were washedtwice with sterile saline and suspended in saline. 100 μl of the cellsuspension was spread onto agar plates containing LBMCG containing 5μg/ml of Tc, and then the plates were incubated for 2 days at 30° C. Thecells grown on the plates were recovered in 7 ml of sterile saline, andthe cell suspensions were serially diluted 10⁻¹-10⁻⁷ in sterile salineand spread onto the same agar plates. To isolate amino acid requiringmutants, the growth of 1,136 colonies grown on LBMCG containing 5 μg/mlof Tc were tested on agar of a minimum medium (referred to as MMhereinafter) composed of 1% glucose, 0.22% NH₄Cl, 0.06% K₂HPO₄, 0.06%KH₂PO₄, 0.04% MgSO₄.7H₂O, 0.02% CaCl₂.2H₂O, 0.04% NaCl, and thefollowing metal salts, vitamins, and nucleic acids (per liter): 12 mg ofFeCl₃.6H₂O, 4 mg of MnSO₄.5H₂O, 0.5 mg of H₃BO₃, 0.4 mg of Na₂MoO₄, 0.32mg of ZnSO₄.7H₂O, 0.04 mg of CuSO₄.5H₂O, 0.002 mg of CoCl₂.6H₂O, 4 mg ofcalcium pantothenate, 3 mg of thiamin-HCl, 1.25 mg of riboflavine, 0.04mg of biotin, 10 mg of hypoxanthine, 10 mg of guanine sulfate, 10 mg ofthymine, and 10 mg of uracil containing 5 μg/ml of Tc. After incubationfor 4 days at 30° C., 37 colonies indicating a poor or no growth werepicked up to LBMCG agar containing 5 μg/ml of Tc, and the productivityof vitamin B₆ was examined by flask fermentation. One loopful cells of37 colonies and S. meliloti IFO14782/pVK601 (parent) were inoculated totubes containing 8 ml of SM medium, and then the tubes were shaken on areciprocal shaker (275 rpm) at 30° C. After shaking for 19 hours, each 4ml of culture broth was transferred to 500-ml flasks with two bafflescontaining 200 ml of PM medium and shaken on a rotary shaker (180 rpm)at 30° C. After cultivation for 7 days, contents of vitamin B₆ in thesupernatant of each culture broth were quantified by HPLC. As a result,S. meliloti PY-C341K1 produced 171 mg of pyridoxol per liter and was1.44 times higher than the parent, strain IFO14782/pVK601.

The amino acid requirement of strain PY-C341K1 together with the parent,strain IFO14782/pVK601, was examined by culturing for 2 days at 30° C.in tubes containing 8 ml of MM supplemented with various kinds of aminoacids. From a result, strain PY-C341K1 grew as well as the parent,IFO14782 /pVK601, in MM medium supplemented with 42 μg/ml of histidine.

EXAMPLE 4

In a similar manner as described in Example 3, S. meliloti PY-C341K1 wascultured in a flask containing LBMCG containing 10 μg/ml of Tc for 16hours at 30° C., and the cell suspension of the strain was prepared. Atube containing 5 ml of the reaction mixtures composed of 0, 30, and 50μg/ml of NTG and 1.6×10⁹ cells per ml in 50 mM Tris-HCl buffer (pH 8.0)was incubated with a reciprocal shaking (275 rpm) for 30 min at 30° C.The cells of each reaction mixture were washed twice with sterile salineand suspended in saline. 100 μl of the cell suspension was spread ontoagar plates containing LBMCG containing 10 μg/ml of Tc, and then theplates were incubated for 2-3 days at 30° C. The cells grown on theplates were recovered by suspending in sterile saline. Aftercentrifugation of the suspension, the cell suspension was diluted togive a turbidity of OD₆₀₀=1.6, and finally to 10⁻⁵. Each 100 μl of thediluents was spread onto five agar plates containing LBMCG containing 10μg/ml of Tc and 0, 0.125, 0.15, or 0.175% glycine because 0.15% glycinecompletely inhibited the growth of S. meliloti PY-C341K1 on LBMCG plate,and then the plates were incubated for 4 days at 30° C. Ten coloniestreated with 50 μg/ml of NTG grown on plates LBMCG containing 10 μg/mlof Tc and 0.175% glycine were picked up on LBMCG agar containing 10μg/ml of Tc. After incubation for 2 days at 30° C., the productivity ofvitamin B₆ in ten colonies together with the parent strain (S. melilotiPY-C341K1) was examined by flask fermentation. One loopful cells wasinoculated to tubes containing 8 ml of SM medium, and then the tubeswere shaken on a reciprocal shaker (275 rpm) at 30° C. After shaking for19 hours, each 4 ml of culture broth was transferred to a 500-ml flaskwith two baffles containing 200 ml of PM medium modified to 0.175%NH₄Cl, and shaken on a rotary shaker (180 rpm) at 30° C. After shakingfor 4 days, sterile solution of urea was added to the each flask at0.125%, and the shaking were further continued for 3 days. The contentsof vitamin B₆ in the supernatant of 7-day culture broth were quantifiedby HPLC method as described in Example 3. As a result, S. melilotiPY-EGC1 produced 362 mg of pyridoxol per liter and was about 2.11 timeshigher than strain PY-341K1 (the parent).

EXAMPLE 5

Vitamin B₆ was recovered from the culture broth of S. meliloti PY-EGC1prepared in the same cultural conditions as described in Example 5.Pyridoxol at each purification step and the concentration was followedby HPLC. One liter of the 168 hour-culture broth containing 344 mg/L ofPN was centrifuged at 7,500 rpm for 10 min. The pH of the resultantsupernatant was adjusted to 3.1 with 1N hydrochloric acid, and then thesupernatant was applied to a column (5.5×15 cm) packed with 350 ml ofAmberlite CG 120 (H⁺ form, 100-200 mesh, Rohm and Haas Company,Philadelphia, Pa., USA). The column was washed with 500 ml of deionizedwater and then eluted with 5% ammonium hydroxide. The vitamin B₆fractions were concentrated under reduced pressure. The residue thusobtained was dissolved in 10 ml of deionized water, and the solution wascharged on a column (5.5×16 cm) packed with 380 ml of Dowex 1×4 (OH³¹form, 200-400 mesh, Dow Chemical Co., Ltd., Midland, Mich., U.S.A.), andthen washed with 500 ml of deionized water. The column was then elutedwith 0.1 N HCl. The fractions containing pyridoxol was concentrated tosmall volume under reduced pressure. After the solid residue wasdissolved in small amount of hot ethanol, the solution was kept standingat 4° C. overnight. The resultant precipitates were collected byfiltration and dried in vacuo to obtain 282 mg of crude crystals. It wasrecrystallized from ethanol to obtain 217 mg of white crystals having amelting point of 160° C. The infrared absorption, UV absorption, and NMRspectrum of the product of the product coincided with those of authenticpyridoxol.

Table 1 summarizes the vitamin B₆ productivities of S. meliloti IFO14782 (DSM No. 10226), S. meliloti IFO 14782/pVK601, and their mutantsso far obtained.

TABLE 1 Pyridoxol Productivities of S. meliloti IFO 14782 (DSM No.10226), S. meliloti IFO 14782/pVK601, and their Mutants PhenotypicPyridoxol Magnitude of Example Microorganism properties (mg/L)increasing S. meliloti IFO 14782 89 1.0 (DSM 10226) 2 S. meliloti IFO14782/ amplification of pdxJ 119 1.34 pVKP601 3 S. meliloti PY-C341K1histidine requirement 171 1.92 4 S. meliloti PY-EGC1 glycine resistance362 4.07

1. A mutant of a recombinant microorganism selected from the groupconsisting of Sinorhizobium meliloti IFO 14782/pVK601, Sinorhizobiummeliloti PY-C341-K1, and Sinorhizobium meliloti PY-EGC1 capable ofproducing vitamin B₆ having a plasmid expressing a recombinant pyridoxol5′-phosphate synthase polypeptide, said plasmid being selected from thegroup consisting of pVK100, pRK290, pLAFR1, and RSF1010 whereby therecombinant microorganism has acquired a phenotypic property ofhistidine requirement or glycine resistance, or a combination of thephenotypic properties thereof.
 2. The mutant of a recombinantmicroorganism according to claim 1, wherein a polynucleotide sequenceencoding said pyridoxol 5′-phosphate synthase polypeptide is cloned intoplasmid pVK100.
 3. The mutant of a recombinant microorganism accordingto claim 1 which is Sinorhizobium meliloti PY-EGC1.
 4. A process forproducing vitamin B₆ which comprises cultivating the mutant according toclaim 1 in a cultivation medium at a pH value of about 5.0 to 9.0, at atemperature of 10° C. to 40° C., and for 1 day to 15 days under aerobicconditions, isolating vitamin B₆ from the cultivation medium.
 5. Theprocess according to claim 4, wherein the mutant is Sinorhizobiummeliloti PY-EGC1.
 6. The mutant of a recombinant microorganism accordingto claim 2, wherein a recombinant plasmid comprising the pyridoxol5′-phosphate synthase gene is pVK601.